Reversed-phase chromatography requires the use of a separation medium that contains hydrophobic alkyl groups as contact sites on the medium surface. The medium itself is a polymer generally formed by polymerization in an aqueous solution, and the inclusion of the hydrophobic alkyl groups requires a further reaction in a nonaqueous medium on the surface of the polymer itself after the polymer has been formed. In large-scale production or in laboratory situations where a large number of analyses or columns are needed, the time required for a second reaction can affect cost and efficiency. Furthermore, when the polymer is porous, penetration of the pores becomes a factor in both the rate of reaction and the uniformity of distribution of the hydrophobic groups on the polymer surface.
Reversed-phase chromatography can be performed in capillaries as well as relatively large-diameter tubing. The use of a capillary permits separations to be performed at high voltage with the rapid dissipation of heat to avoid excessive joule heating. Further advantages are the use of very small amounts of sample, and the completion of an analysis in a relatively short period of time. A reversed-phase separation however requires that the capillary be packed with solid phase such as beads, which is difficult to do in a capillary, particularly in a manner that will result in a uniform packing density. Another difficulty in the use of a packed capillary is that a supporting frit is generally required at the capillary outlet to hold the beads in place. Preparation of the frit is not a simple task, and the frit often generates air bubbles during the separation. For these reasons, capillaries are not widely used for reversed-phase chromatography.
These disadvantages are eliminated by the formation of a continuous bed in the capillary, i.e., a monolithic porous polymer used in place of the beads, the polymer having been formed by polymerization in the capillary itself, spanning the entire cross section of the capillary and bonded to the capillary wall. A description of this type of bed is found in granted European Patent Specification No. 0 407 560 of Bio-Rad Laboratories, Inc., and its United States counterpart, pending application Ser. No. 08/400,419, filed Mar. 2, 1995. The disclosures of both of these documents are incorporated herein by reference.
Like polymeric separation media in general, however, the formation of a continuous porous polymer bed is performed with an aqueous solution, and the resulting bed is at least primarily hydrophilic. The subsequent reaction to place hydrophobic groups on the polymer surface must be performed inside the capillary, and here again, the need for two reactions adds to the cost of the column.